Cell-based assays that investigate the effect of small molecules on cellular activities are becoming increasingly popular screening tools for drug discovery. The ability to assess small molecules in a physiologically relevant environment is informative, as the assays simultaneously evaluate compound permeability, toxicity, and activity. Yet, cell-based assays are often limited to detect global changes in cellular behavior, such as gene expression or phenotype. The current challenge is to develop cell-based assays for specific enzyme activities. However, the most common strategies for measuring enzyme activities, such as radioactivity, absorbance, and fluorescence labels, often cannot be delivered to the appropriate target within the cellular environment. This proposal describes a method that combines cell culture and lysis on customized surface chemistries that present cell adhesion ligands along with substrates for distinct enzyme activities. The cells are cultured and lysed directly on the monolayers in the presence of substrates that record the activity of enzymes within the lysate. The activity is measured using a high-throughput, mass-spectrometry based readout termed SAMDI (self-assembled monolayers for matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS)). The technique, termed Tandem Culture and Lysis-SAMDI (TCAL- SAMDI) is amenable to any adherent cell line and can measure virtually any enzyme activity. TCAL-SAMDI will offer drug discovery companies in a broad range of diseases a platform for cell-based, high-throughput, and label-free screen of specific enzyme activities. !